Abstract There are three major isoforms of apolipoprotein E (called apoE2, apoE3 and apoE4) that arise from a single gene locus and differ from each other only by single amino acids. It is now well-established that the major risk factor for the development of late-onset Alzheimer's disease (accounting for about 90% of cases of dementia) is the presence of the apoE4 isoform. This protein is so insidious that individuals with two copies of apoE4 will develop Alzheimer's disease by age 70. Our hypothesis is that the difference in the interaction of apoE isoforms with amyloid-b, the major protein in amyloid plaques, is responsible for the functional differences between apoE isoforms. There is considerable evidence that Ab clearance from cells is a defining factor in the development of Alzheimer's disease and is apoE isoform dependent. Our goal is to understand the apoE-Ab interaction. There are three specific aims: To understand the kinetics and mechanism of Ab aggregation, to understand the apoE- Ab and apoE-Ab-lipid interaction and to find small molecules that perturb these interactions. The apoE-Ab interaction is poorly understood. Thus, the Ab binding site on apoE, the forms of Ab that bind apoE, the role of lipid in Ab binding and the mechanism of Ab binding to apoE are largely unknown. We have shown that apoE interacts only with intermediates that occur during Ab aggregation making it is essential to understand the mechanism of Ab aggregation itself. We ask what those intermediates are and how they define the interaction with apoE using new fluorescence assays we have developed that first determine the rate and equilibrium constants for the formation of oligomers during the Ab aggregation and secondly examine the aggregation process beyond oligomer formation. These methods will be then used to understand the role of lipid on the apoE-Ab interaction. We will use our recent observation of structural differences between apoE3 and apoE4, differences distant from the site of the single amino acid change, as the basis for examining the differences in the interaction between apoE isoforms and Ab. Specific regions/residues of both apoE and Ab involved in their interaction will be determined by site-directed mutations and/or hydrogen/deuterium exchange procedures. These methods, along with high throughput screening experiments we propose, will be used to differentiate the behavior of apoE4 relative to that of apoE3 with respect to the interaction with Ab. The results may lead to the new ideas concerning the development of therapeutic agents to delay the onset of Alzheimer's disease. In collaboration with others at Washington University any small molecule compounds that do so will be tested for their ability to affect Ab clearance in mouse brain. Of critical importance is the availability of the structure of a full-length monomeric form of apoE3 (34 kDa), the only known full-length structure of any apoE isoform.